Location of functional -SH groups in NADPH-cytochrome P-450 reductase from rabbit liver microsomes.
The total -SH content of purified NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) from rabbit liver microsomes accessible to an excess equivalent of PCMB was 7.0 +/- 0.3 mol thiol groups/mol protein. The modification of four -SH groups at low concentration of PCMB stimulated the activity of the enzyme. On the other hand, further blocking of -SH groups (6-7 mol -SH groups/Mol protein) with an excess amount of PCMB completely inhibited cytochrome c (or DCPI) reductase activity. The fluorescence quenching of the flavin was rapidly removed by binding of PCMB to a fifth and sixth -SH group during a gradual titration. Kinetic and fluorimetric analyses confirmed the suggestion that these two -SH groups essential for catalytic function were partly protected by NADP+ or 2'-AMP against the reaction with PCMB. Excess PCMB begins to compete with the ligand preincubated with the enzyme. The spectral perturbation on the addition of approx. 6-7 equiv. PCMB/mol enzyme is accompanied by a slight blue shift of the absorbance maximum at 380 nm, with the appearance of a pronounced shoulder at 475 nm. In contrast to the native enzyme, 3-electron-reduced semiquinone form of PCMB-treated enzyme showed the same absorption spectrum as 1-electron-reduced semiquinone which has an absorption maximum at 585 nm with a broad shoulder around 635 nm. An inhibitory effect may be attributable to the fact that NADPH is less accessible to the FAD binding site as well as the pyridine nucleotide binding site, since the rate of FAD reduction becomes extremely slow after complete modification.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000693:Anaerobiosis; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology*; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D008024:Ligands; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase